Intestinal Microbiota Affect Stroke Outcome by Modulating the Dendritic Cell-regulatory T Cell Axis

项目总结 在美国，中风是一种毁灭性的疾病，也是导致死亡和残疾的主要原因。缺血性卒中 导致大量免疫细胞的大量激活，这些免疫细胞可以在血脑屏障之后渗透到大脑中 崩溃了。肠道微生物区系此前已被认为是影响预后的重要因素 在临床研究和动物模型中缺血性卒中的严重性。然而，潜在的机制 微生物区系对中风后免疫细胞的调节作用尚不清楚。树突状细胞(DC)充当 先天免疫和获得性免疫之间的桥梁，因为它们能够从肠腔和 塑造T细胞反应。抗生素诱导的小鼠微生物区系改变导致卒中神经保护作用 小鼠大脑中动脉闭塞后缺血性卒中模型与对照组的比较 常规微生物区系，经过类似处理，但携带抗药性微生物区系，导致 微生物区系类似于幼稚的小鼠。这种影响归因于肠道和肠系膜的较大容量。 携带“改变的”微生物区系的小鼠淋巴结树突状细胞在小鼠体内诱导T调节细胞(Treg) 肠道随后抑制流向大脑的破坏性促炎IL-17+γδT细胞 中风后。使用我们的体外模型来模拟肠道DC-T细胞的相互作用，我们证明了 从携带“改变的”或“常规的”微生物区系的小鼠的小肠(SIC)分离的内容物的DC 随后与CD4细胞共培养类似地诱导了小鼠SIC后更大比例的Tregs 携带“改变的”微生物区系，而不是来自具有“常规”微生物区系的小鼠的碳化硅。这项建议旨在 阐明改变微生物区系可能导致模式识别受体或 Toll样受体配体负责DC耐受表型和Treg诱导 小鼠的微生物区系改变。使用各种体外和体内方法，我的目标是识别DC受体 以及负责感知这些腔内容物并产生耐受性表型的信号机制， 确定DC产生的信号/细胞因子是肠道Treg诱导所必需的，并确定PRO-2的作用。 携带“改变”或“常规”基因的小鼠卒中神经保护中炎性IL-6与不良卒中结局的比较 微生物区系。总之，我试图理解肠道DC受体配体是如何依赖微生物区系的 可作为肠道免疫和中风预后的调节器，以及确定潜在的治疗靶点。

PROJECT SUMMARY Stroke is a devastating disease and leading cause of death and disability in the United States. Ischemic stroke results in massive activation of numerous immune cells that can infiltrate the brain following blood-brain-barrier breakdown. The gut microbiota has previously been identified as a significant factor affecting outcome and severity of ischemic stroke in clinical studies and animal models. However, mechanisms underlying the modulatory role of microbiota on immune cells following stroke remain unclear. Dendritic cells (DCs) act as the bridge between innate and adaptive immunity, with their ability to sample material from the intestinal lumen and shape T-cell responses. Antibiotic-induced alteration of microbiota in mice results in stroke neuroprotection in mice following middle cerebral artery occlusion model of ischemic stroke compared to control mice carrying conventional microbiota, which are similarly treated but carry antibiotic-resistant microbiota resulting in microbiota similar to that of naïve mice. This effect is attributed to the greater capacity of intestinal and mesenteric lymph node dendritic cells of mice carrying “altered” microbiota to induce T-regulatory cells (Tregs) in the small intestine which subsequently suppress destructive pro-inflammatory IL-17+ γδ T cells that traffic to the brain following stroke. Using our in vitro model to simulate intestinal DC-T cell interactions, we show that priming naïve DCs with isolated contents from the small intestine (SIC) of mice carrying “altered” or “conventional” microbiota and subsequent co-culture with CD4 cells similarly induces greater proportions of Tregs following SIC from mice carrying “altered” microbiota compared to SIC from mice with “conventional” microbiota. This proposal seeks to elucidate the mechanism by which altering microbiota may result in changes in pattern-recognition receptors or toll-like receptor ligands that are responsible for a DC-tolerizing phenotype and Treg induction observed with microbiota alteration in mice. Using a variety of in vitro and in vivo approaches, I aim to identify the DC receptors and signaling machinery responsible for sensing these luminal contents and producing a tolerogenic phenotype, determine DC-produced signals/cytokines necessary for intestinal Treg induction, and establish the role of pro- inflammatory IL-6 in stroke neuroprotection vs poor stroke outcome in mice carrying “altered” or “conventional” microbiota. In summary, I seek to understand how intestinal DC receptor ligands that are microbiota-dependent can act as regulators of intestinal immunity and stroke outcome, as well as identify potential therapeutic targets.